PROJECT SUMMARY/ABSTRACT The relative scarcity of recurrent somatic mutations in neuroblastoma, a tumor of the developing sympathetic nervous system, has challenged efforts to devise effective targeted therapies for this devastating childhood cancer. One exception to this otherwise disappointing picture is the mutated ALK receptor tyrosine kinase, which appears at a relatively high frequency in neuroblastoma, and has been highlighted as an important player in the pathogenesis of this tumor and a key target for drug development. Research progress over the past 5 years in the principal investigator?s (PI?s) laboratory (under 1R01CA148688) and recently acquired preliminary data indicate that targeted therapies combining improved ALK inhibitors (ALKi) with novel synergistic inhibitory compounds are needed to induce durable remissions in neuroblastoma patients with activating ALK mutations. This projected advance will need to be coupled with greater knowledge of the origins of resistance to such treatment, as well as strategies to extend ALK-directed therapy to larger numbers of patients. To address these challenges, the PI will pursue three pivotal questions that are intended to propel targeted therapy research in neuroblastoma into the next era ? the long-term goal of this research. Aim 1: Can the cytotoxicity of ALKi be improved and resistance delayed by combining these agents with transcriptional CDK inhibitors (CDKi)? Hypothesis ? The combination of CDKi with ALKi in ALK-mutated, MYCN nonamplified neuroblastoma cells produces synergistic activity by disrupting transcriptional regulation through enhanced inhibition of ALK signaling. Aim 2: What are the mechanisms by which ALK-mutated, MYCN-amplified neuroblastoma cells develop resistance to ALK inhibitors? Hypothesis ? The acquisition of resistance to ALKi is associated with global redistribution of super-enhancers to BRD4 enhancer sites, leading to novel molecular vulnerabilities that could be exploited for therapeutic gain. Aim 3: Can wild-type ALK cleavage be leveraged as a therapeutic target in high-risk neuroblastoma? Hypothesis ? Proteolytic cleavage of the extracellular domain of ALK could be therapeutically targeted through MMP9 inhibition to impede NB cell migration. Positive results in this renewal proposal would clearly benefit children with high-risk neuroblastoma and may also benefit patients with different cancers that express aberrant ALK, from non-small cell lung cancer to anaplastic large-cell lymphoma.